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Determination of acrylamide in dry feedstuff for dogs and cats

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Acrylamide is considered to be an endogenous contaminant of food and feedstuff. Attention is paid to the acrylamide content in human nutrition products; however, there is lack of data about its concentrations in feedstuff. The aim of this study was to use a newly developed adsorptive stripping voltammetry procedure for determination of acrylamide concentrations in five and three different kinds of dog and cat dry feedstuff, respectively. The applied analytical procedure consists of a solvent extraction in ultrasound bath, followed by voltammetric measurement at the hanging mercury drop electrode in ammonia buffer. The accuracy of the method was verified by use of standard reference materials. The range of acrylamide concentration found in samples of dry dog and cat feedstuff ranged from 106 to 358 μg/kg, and from 66 to 269 μg/kg, respectively. The precision of analyses expressed in form of the relative standard deviations ranged between 0.6-1.7%. The voltammetric procedure appears to be a reliable, sensitive, rapid and low-cost analytical technique for the determination of acrylamide in food and feedstuff. The concentrations of acrylamide found in feedstuff were relatively moderate but it is undoubtedly necessary to monitor its concentrations in future.
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Determination of acrylamide in dry feedstuff for dogs and cats
Helena Veselá, Emanuel Šucman
University of Veterinary and Pharmaceutical Sciences Brno, Faculty of Veterinary Hygiene and Ecology,
Institute of Biochemistry, Chemistry and Biophysics, Brno, Czech Republic
Received September 4, 2012
Accepted March 19, 2013
Abstract
Acrylamide is considered to be an endogenous contaminant of food and feedstuff. Attention is
paid to the acrylamide content in human nutrition products; however, there is lack of data about
its concentrations in feedstuff. The aim of this study was to use a newly developed adsorptive
stripping voltammetry procedure for determination of acrylamide concentrations in ve and
three different kinds of dog and cat dry feedstuff, respectively. The applied analytical procedure
consists of a solvent extraction in ultrasound bath, followed by voltammetric measurement at the
hanging mercury drop electrode in ammonia buffer. The accuracy of the method was veried by
use of standard reference materials. The range of acrylamide concentration found in samples of
dry dog and cat feedstuff ranged from 106 to 358 μg/kg, and from 66 to 269 μg/kg, respectively.
The precision of analyses expressed in form of the relative standard deviations ranged between
0.6–1.7%. The voltammetric procedure appears to be a reliable, sensitive, rapid and low-cost
analytical technique for the determination of acrylamide in food and feedstuff. The concentrations
of acrylamide found in feedstuff were relatively moderate but it is undoubtedly necessary to
monitor its concentrations in future.
Dry dog feed, dry cat feed, endogenous contaminant, analysis, voltammetry
Acrylamide (AA) is a commercially produced substance. It can be naturally formed at
high temperatures in food/feedstuff rich in starch. The major pathway of AA formation
is a reaction between an amino acid and a carbonyl group of reducing sugar, known as
Maillard reaction. Acrylamide can be absorbed by animals and humans via ingestion,
inhalation or through the skin. Regardless of the route of absorption, AA is distributed
relatively rapidly to all tissues. It was found in human tissues such as in the thymus, liver,
heart, brain, kidneys, placenta and breast milk that the major metabolite glycidamide is
formed due to oxidation of AA by cytochrome P450. Acrylamide and glycidamide can
bind in vivo haemoglobin, serum albumins, DNA, and enzymes. Acrylamide affects both
humans and animals. Typical symptoms of acrylamide exposure include ataxia and skeletal
muscle weakness (Capuano and Fogliano 2011). The International Agency for Research
on Cancer (IARC 1997) has classied acrylamide in Group 2A (probable carcinogen to
humans). Acrylamide is a multi-organ carcinogen that causes tumours of lungs, uterus,
mammary gland etc. It was conrmed that AA causes cancer in mice and rats (Törnquist
2005). Genetic toxicity and reproductive toxicity have been proven in a variety of biological
assays (Von Mühlendahl and Otto 2003; Törnquist 2005; Capuano and Fogliano
2011). A comprehensive detailed overview of acrylamide contents in different kinds of
food and of methods available for its determination is given in the EFSA Report (2012).
Analytical methods suitable for the determination of acrylamide in food and/or feedstuff
have been explored. Gas chromatography (GC), high performance liquid chromatography
(HPLC) and capillary electrophoresis (CE) have been used for determination of acrylamide
in food being predisposed to acrylamide formation. The technique of HPLC equipped with
mass spectrometry (MS) detector is most often used (Keramat 2001; Zhang et al. 2005;
Tekkeli et al. 2012). Electrochemical methods are widely used in environmental and/or
ACTA VET. BRNO 2013, 82: 203–208; doi:10.2754/avb201382020203
Address for correspondence:
Prof. RNDr. Emanuel Šucman, CSc.
Institute of Biochemistry, Chemistry and Biophysics
University of Veterinary and Pharmaceutical Sciences Brno
Palackého tř. 1/3, 612 42 Brno
Czech Republic
Phone: +420 541 562 603
E-mail: sucmane@vfu.cz
http://actavet.vfu.cz/
nutritional assessments. The advantage of the electrochemical approach is a relatively short
measuring time including sample preparation (Wang 2001). Amounts of acrylamide were
measured by square-wave voltammetry in potato chips (Zargar et al. 2009). Recently, a
new approach to electrochemical determination of AA was published (Veselá and Šucman
2013). In this study the use of adsorptive differential pulse voltammetry for determination
and quantication of the AA content in food and feedstuff was described. The analytical
properties of this newly developed electroanalytical method are described as well as its
comparison with common chromatographic methods, especially the HPLC techniques. In
the available literature data on the content of AA in feedstuffs are absent. Nevertheless,
their composition and/or technological processes make possible the occurrence of AA in
this material.
The aim of this study was to check the usefulness of the new electrochemical method to
analyse the amounts of acrylamide in dry dog and cat feedstuffs.
Material and Methods
Material for analysis
The content of acrylamide was measured in ve randomly selected dry dog feedstuffs purchased from local
markets: Friskies junior dry, Purina (Nestlé Česko s.r.o.), Chappi dry dog foods meat and vegetables (Mars
Czech s.r.o.), Propesko complete menu for small and medium breeds (ProvimiPet Food CZ s.r.o.), Darling with
meat and added selected vegetables, Purina (Nestlé Česko s.r.o.) and Butcher’s dry complete beef (Butchers
Pet Care Ltd, UK). Furthermore, three randomly selected dry cat foodstuffs were purchased: Friskies indoor
cats, Purina (Nestlé Česko s.r.o.), Kitekat dry beef vegetables (Mars Czech s.r.o) and Perfect t sensitive (Mars
Czech s.r.o.). All the feedstuffs are registered for use in the Czech Republic and their composition complies with
assessed ranges. Five grams of each feedstuff sample were taken for analysis. Rusk (European Reference Material
ERM
®
-BD274, Germany) and crispbread (European Reference Material ERM
®
-BD272, Germany) were used as
the Standard Reference Materials (SRM) in order to check the accuracy of analysis. The other alternative was the
use of recovery measurements because none SRM for feedstuff exist.
Chemicals
Mercury (polarographic grade) and acrylamide (electrophoresis grade) were supplied by Merck (Germany).
Nickel solution Astasol (Analytika, Czech Republic) was used (1.000 ± 0.005 g/l). In order to remove dissolved
oxygen from solutions Argon (Linde Technoplyn a.s., Czech Republic) was used. All chemicals used for samples
and/or standard preparation were of analytical grade (Merck, Germany). Ultra-pure water (GenPure, TKA GmbH,
Germany) of specic conductivity < 1 μS·m
-1
was used to prepare all solutions.
Equipment
Measurement of acrylamide concentration was performed by the Autolab measuring unit (Eco Chemie B.V.,
Netherlands) equipped with the electrode system VA Stand 663 (Metrohm, Switzerland). The software Nova 1.5
(Eco Chemie B.V., Netherlands) was used for measurement control and evaluation. The hanging mercury drop
electrode was the working electrode; the Ag/AgCl was the reference electrode (3 mol/l KCl), and the glassy
carbon electrode was used as the auxiliary electrode.
Procedure
The principle of the method is known as the adsorptive stripping voltammetry which is based upon formation
of a complex compound of AA with Ni
2+
. Such complex can be adsorbed on the surface of the mercury working
electrode and measured after sufcient accumulation time. The parameters of the measurement procedure had
been thoroughly evaluated
in order to nd the set of optimal conditions. The exact analytical procedure was
described in article by Veselá and Šucman (2013).
The statistical methods used for data evaluation are described in Eckschlager et al. (1980).
Results
In both SRMs and samples the peak heights were found to be a function of acrylamide
concentration and/or deposition time. Measurement parameters were thoroughly set in
order to get appropriate results. A linear relationship between currents of peak maxima and
concentration of AA was found. The detection limit of the method was 12 µg/kg under the
selected measurement conditions.
204
Samples of standard reference materials (Rusk BD-272 and Crispbread BD-274) were
analysed in order to conrm the accuracy of the applied method. Additionally, the accuracy
was also checked in real samples using the standard addition approach. An example of
voltammogram and its evaluation is given in the Figs 1 and 2. The linear relationship
between peak currents and concentrations is clearly seen. The measured values were
205
9E-8
8E-8
7E-8
6E-8
5E-8
4E-8
3E-8
2E-8
1E-8
0
-0.6 -0.55 -0.5 -0.45 -0.4 -0.35 -0.3 -0.25 -0.2 -0.15 -0.1
Potential applied (V)
WE(1).o.Current (A)
-
6.00E-08
5.00E-08
4.00E-08
3.00E-08
2.00E-08
1.00E-08
0.00E
+
08
m (ng)
y = 1.04E-08x + 1.66E-08
R
2
= 9.96E-01
0 0.5 1 1.5 2 2.5 3 3.5
I (A)
Fig. 1. Voltammetric curves of acrylamide in the standard reference material rusk
E
d
= -0.72 V, E
e
= -0.05 V, SR=5 mV/s, PH=50 mV, T
d
=300 s. Curves: 1 sample (200 µl), 2 – standard addition
(1.56 ng of acrylamide), 3 – standard addition (3.13 ng of acrylamide)
E
d
–deposition potential (V), E
e
–nal potential (V), SR–scan rate (V/s), PH–peak height (mV), T
d
–deposition
time (s).
Fig. 2. Dependence of peak heights on the concentration of acrylamide in the standard reference material rusk
m – amount of added acrylamide (ng), I – measured current (A)
77 ± 10 µg/kg and 992 ± 113 µg/kg for rusk and crispbread, respectively. Concentrations
of acrylamide found in these SRMs are in good agreement with the data specied in their
certicates (Table 1).
The results of AA determination in dog dry feedstuff are given in Table 2. Acrylamide was
found at a concentration ranging from 106 to 358 μg/kg. The relative standard deviation
ranged from 0.6 to 1.7%. The calculated recoveries with known standard additions of AA
(Table 3) for this feedstuff are close to the required 100% (98.7–99.8%); no signicant
deviation was found.
The results found in dry cat foodstuff are given in the Table 4. Acrylamide was found
206
Table 1. Concentrations of acrylamide in standard reference materials.
n – number of samples, CI – 95% condence interval, s – standard deviation, RSD – relative standard deviation
Sample n Certied (μg/kg) 95% CI (μg/kg) s (μg/kg) RSD (%)
Rusk BD-272 3 72 ± 4 77 ± 10 4.7 6.1
Crispbread BD-274 4 980 ± 90 992 ± 113 92.8 9.3
Table 2. Concentrations of acrylamide in dry dog feedstuff.
n – number of samples, 95%CI – condence interval, s – standard deviation, RSD – relative standard deviation
Sample n 95% CI (μg/kg) s (μg/kg) RSD (%)
Friskies 3 214 ± 3 1.2 0.6
Chappi 3 106 ± 3 1.2 1.1
Propesko 3 117 ± 4 1.8 1.5
Darling 3 358 ± 13 5.9 1.7
Butchers 3 222 ± 7 3 1.3
Table 4. Concentrations of acrylamide in dry cat feedstuff.
n – number of samples, 95%CI – condence interval, s – standard deviation, RSD – relative standard deviation
Sample n 95% CI (μg/kg) s (μg/kg) RSD (%)
Friskies 3 269 ± 7 3 1.1
Kitekat 3 103 ± 4 1.8 1.7
Perfect Fit 3 66 ± 1 0.6 0.9
Table 3. Accuracy checking in samples of dry dog feedstuff using acrylamide standard addition.
n – number of samples, 95%CI – condence interval, AA – acrylamide
Sample n 95% CI (μg/kg) Added AA (μg/kg) Recovery (%)
Friskies 3 427 ± 5 214 99.8
Chappi 3 210 ± 4 106 99.1
Propesko 3 231 ± 5 117 98.7
Darling 3 715 ± 12 358 99.9
Butcher‘s 3 439 ± 6 222 98.9
at a concentration ranging from 66 to 269 μg/kg. Relative standard deviation of results
ranged from 0.9 to 1.1%. The calculated recoveries of added AA standard (Table 5) in this
feedstuff were close to the required 100% (99–100%); no signicant deviation was found.
This gives the evidence for the adequate accuracy of the applied voltammetric method for
determination of AA in feedstuff.
Discussion
There is a lack of data about acrylamide concentrations in dog and cat dry feedstuff in
the available literature. This study introduces a new way for acrylamide determination in
feedstuff. The voltammetric procedure seems to be a reliable, sensitive, rapid and low-cost
analytical technique for determination of acrylamide in the above mentioned feedstuff.
The total analysis time including the extraction step and measurement evaluation was
approximately 2 h. The measured and calculated recoveries of acrylamide both in dog and
in cat dry food as well as the results measured in reference materials proved the adequate
accuracy of the method. The precision expressed in terms of relative standard deviations
ranged from 0.6 to 1.7% which is satisfactory for this kind of analysis.
The total analysis time including the extraction step and measurement evaluation was
approximately 2 h. The concentrations found were relatively moderate compared to
human food. Nevertheless, there is no doubt that acrylamide, relatively new carcinogenic
endogenous contaminant, is present also in these feedstuffs. Therefore it is necessary to
monitor its concentrations.
Acknowledgements
This research was supported by the Ministry of Education, Youth and Physical Training of the Czech Republic
(Research Plan Veterinary Aspects of Food Quality and Safety, MSM 6215712402).
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Table 5. Accuracy checking in samples of dry cat feedstuff using acrylamide standard addition.
n – number of samples, 95%CI – condence interval, AA – acrylamide
Sample n 95% CI (μg/kg) Added AA (μg/kg) Recovery (%)
Friskies 3 533 ± 4 269 99.1
Kitekat 3 204 ± 5 103 99
Perfect Fit 3 132 ± 7 66 100
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review and recent developments. J Chromatogr A 1075: 1-21
208
... The regurgitated material initially appears undigested and is covered with mucus. It is difficult to clearly define the onset of regurgitation, and it is reported that it starts on the 54-68 days following the AA application [24]. ...
... When research on the AA concentrations of dog and cat foods is examined, it is observed that there are very few sources on the subject. Veselá and Šucman [24] studied the AA level in dry dog and cat food from various companies by the voltammetric method. To test the accuracy of the method, the authors used rusk and crispbread in the control group. ...
... In addition, only 2 publications with similar methods were found by the same authors on the determination of AA in cat and dog foods. In both of these studies, AA levels were measured voltammetrically and similar results were reported [24][25]. The authors stated that the use of voltammetric methods in the determination of AA in dry cat and dog foods is reliable, sensitive, fast, and cost-effective; they also calculated the total time spent for analysis, including extraction and evaluation of measurements, as approximately 2 h. ...
... Acrylamide exposure arises from cigarette smoke (Urban et al., 2006), as well as processed or 376 heated food products such as potatoes, cereals, coffee, crackers or breads, and dry pet foods 377 (Vesela and Sucman, 2013;Tareke et al., 2002). Maximum allowable concentrations of acrylamide 378 in pet foods have not been formulated. ...
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